Chitosan manufacturing process

ABSTRACT

A method for producing chitosan from naturally occurring chitin-containing raw material, such as crustacean shells, includes an optional pretreatment step to remove non-chitin rich organic material for example, shrimp flesh, from the raw material, e.g., shrimp shells. The optional pre-treatment is followed by a demineralization step utilizing a mild hydrochloric acid solution and a deproteination step utilizing a mild sodium hydroxide solution. The deproteination step is followed by a deacetylation step to remove the acetyl group from N-acetylglucosamine (chitin) to form an amine group, yielding d-glucosamine (chitosan). Each step is followed by a washing step and the product is dried, preferably at a temperature not in excess of about 65° C. Known purification and grinding steps may also be used to produce the final chitosan product. The process is carried out in equipment comprising a series of substantially identical or similar tanks ( 18, 26, 36,  etc.) and dryers ( 62, 62 ′), suitably interconnected.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of patent applicationSer. No. 13/684,687, filed on Nov. 26, 2012, which is a divisionalapplication of patent application Ser. No. 12/406,476, filed on Mar. 18,2009, now U.S. Pat. No. 8,318,913, which claims the benefit of priorityof the filing date of provisional patent application Ser. No.61/037,742, filed on Mar. 19, 2008, all entitled “CHITOSAN MANUFACTURINGPROCESS”.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns a method for the production of chitosanfrom naturally occurring chitin-containing materials.

2. Related Art

Chitin (C₈H₁₃NO₅)n is a naturally occurring N-acetylglucosaminepolysaccharide that is obtainable from a variety of sources, especiallyexoskeletons of marine animals; for example, chitin is a principalcomponent of the shells of crustaceans. See the article by Mathur, N. K.and Narang, C. K.; “Chitin and chitosan, versatile polysaccharides frommarine animals”; Journal of Chemical Education; v. 67, 1990, p. 938, thedisclosure of which is incorporated by reference herein.

The following documents are typical of many which disclose variousschemes for production of chitosan; Patent Publication US 2006/0205932A1, Patent Publication CN 1371922A, Patent Publication CN 1158335A,Patent Publication CN 101177328A, and U.S. Pat. No. 4,066,735.

SUMMARY OF THE INVENTION

Generally, the process of the present invention comprises a process tomanufacture chitosan comprising the steps of: pretreatment (not neededin all cases), demineralization, deproteination, deacetylation anddrying, with water washes after each step except, of course, the dryingand dewatering steps. The steps of the process are preferably carriedout in the order listed. The starting material may be any naturallyoccurring source of chitin, such as shells of crustaceans, for example,waste shrimp shells resulting from processing of shrimp. The process ofthe invention provides a white medical-grade quality chitosan withoutneed for any of the prior art chitosan decolorizing steps.

More specifically, in accordance with the present invention there isprovided a process for the manufacture of chitosan from a naturallyoccurring chitin source, the process comprising the following steps. Anaturally occurring chitin source is demineralized by immersing it in ademineralization (sometimes, “DMIN”) hydrochloric acid solution,preferably of from about 0.5 to about 2 Molar (M) HCl, more preferablyfrom about 0.9 to about 1.1 M, at a temperature of from about 20° C. toabout 30° C., more preferably from about 22° C. to about 26° C., and fora DMIN period, preferably of about 0.5 to about 2 hours, more preferablyfrom about 0.75 to about 1.25 hours, and then separating the resultingdemineralized chitin source from the acid solution, washing the chitinsource in a DMIN wash water for a DMIN wash period, preferably of about0.5 to about 2 hours, more preferably from about 0.9 to about 1.1 hours,and then separating the demineralized chitin source from the DMIN washwater. The demineralized chitin source is subjected to deproteination(sometimes, “DPRO”) by treating the demineralized chitin source in aDPRO sodium hydroxide solution preferably containing from about 1% toabout 10% w/v NaOH, more preferably from about 4% to about 6%, at atemperature preferably from about 60° C. to about 80° C., morepreferably from about 70° C. to about 75° C., for a DPRO period,preferably of about 4 to about 24 hours, more preferably from about 4 toabout 6 hours, and then separating the resulting DMIN and DPRO chitinsource from the deproteination sodium hydroxide solution, washing theseparated DMIN and DPRO chitin source in a DPRO wash water, preferablyfor a DPRO wash period of from about 0.5 to about 2 hours, morepreferably for about 1 hour, and then separating the DMIN and DPROchitin source from the deproteination wash water. Residual water is thenseparated from the DMIN and DPRO chitin. The chitin source obtained fromthe deproteination step is immersed into a concentrated sodium hydroxidedeacetylation (sometimes, “DEAC”) solution preferably containing fromabout 40% to about 50% w/w NaOH, more preferably from about 45% to about50% w/w, at a temperature of from about 90° C. to about 110° C. for aDEAC period of time sufficient to convert acetyl groups of the chitinsource obtained from the deproteination step to amine groups, to therebyform a chitosan biopolymer having d-glucosamine as the monomer of thechitin biopolymer. The resulting chitosan biopolymer is separated fromthe DEAC solution and the separated chitosan biopolymer is washed in aDEAC wash water, preferably for a DEAC wash period of from about 1 toabout 3 hours, more preferably from about 0.9 to about 1.1 hours, andthen separating the chitosan biopolymer from the DEAC wash water.Residual water is then separated from the chitosan biopolymer which isthen dried in air, preferably at a temperature of about 50° C. to about65° C., more preferably, from about 50° C. to about 60° C., for a timeperiod preferably from about 4 to about 6 hours, more preferably fromabout 2 to about 5 hours, to reduce the moisture content of the chitosanbiopolymer to below 10% to provide a medical-grade quality chitosan.

In another aspect of the present invention there is provided in theabove process an optional, initial pretreatment (sometimes, “PTRT”) stepin order to remove non-chitin rich organic material from the naturallyoccurring chitin source by treating the chitin source with a mildpre-treatment sodium hydroxide solution, preferably containing fromabout 1% to about 4% w/v NaOH for about 2 to about 24 hours at atemperature, preferably of from about 20° C. to about 30° C., to removenon-chitin organic material. The resulting pretreated chitin source isseparated from the pretreatment sodium hydroxide solution, and then thepretreated chitin source is washed for a PTRT wash period of, preferablyfrom about 0.5 to about 2 hours.

Other aspects of the present invention provide one or more of thefollowing steps alone or in any suitable combination. The naturallyoccurring chitin source may comprise exoskeletons of a marine animal;the naturally occurring chitin source may comprise crustacean shells;the naturally occurring chitin source may comprise shrimp shells; thedemineralization step may be carried out before the deproteination step;the chitosan polymer obtained from the deacetylation step is in flakeform and is thereafter ground into powder form; one or more additionalsteps of the process may be provided, the additional steps consistingessentially of removing foreign particles, arsenic, mercury, lead andother heavy metals, and microbiological contaminants from the chitinsource at any step of its treatment and any of the materials resultingfrom treatment of the chitin source; any additional process stepsdirected at whitening the chitosan product other than those defined inany or all of the process steps above, may be excluded.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a legend showing that FIG. 1B is a continuation of FIG. 1A;

FIG. 2 is a legend showing that FIG. 2B is a continuation of FIG. 2A;

FIG. 1A is a schematic plan view of the first part of a manufacturingline for the extraction of chitin from shrimp shells in accordance withan embodiment of the present invention;

FIG. 1B is a schematic plan view of the second part of the manufacturingline, the first part of which is shown in FIG. 1B;

FIG. 2A is a schematic plan view of a manufacturing line for theconversion of chitin to chitosan in accordance with an embodiment of thepresent invention;

FIG. 2B is a schematic plan view of the second part of the manufacturingline, the first part of which is shown in FIG. 2A;

FIG. 3 is a schematic, cross-sectional elevation view of a typicalprocess tank used in the manufacturing lines of FIGS. 1 and 2;

FIG. 3A is a schematic plan view of the process tank of FIG. 3; and

FIG. 3B is a schematic, cross-sectional elevation view of the processtank of FIG. 3 rotated ninety degrees to counterclockwise from itsposition in FIG. 3A of the drawings.

DETAILED DESCRIPTION OF THE INVENTION AND SPECIFIC EMBODIMENTS THEREOF

All steps of the process of the present invention may conveniently beperformed in a series of substantially uniform cylindricaltank/screen/mixer arrangements of the type shown in FIGS. 3, 3A and 3Bof the drawings. Substantial uniformity of the several reactor vesselsrequired provides economies in initial capital investment and inmaintenance and repair. Numerous pumps are shown in the drawings but arenot numbered or specifically noted, as their function will be clear tothose skilled in the art from the location of the pumps and thedescription of the process. The water used for all post-treatment washesdescribed below is at the temperature at which unheated water issupplied to the facility in which the process is being carried out.

Pretreatment Step

The pretreatment step is optional in the sense that it is needed only ifthe chitin source contains a significant amount of non-chitin richorganic material. (The term “non-chitin rich organic material” includesmaterial containing no chitin.) In such cases, the pretreatment step isused to remove non-chitin rich organic material from the organic sourceof chitin, for example, to remove residual shrimp flesh from shrimpshells. The following description refers to shrimp shells as thenaturally occurring source of chitin although it will be appreciatedthat other marine life exoskeletons, that is, shells, especially shellsof crustaceans are major sources of natural chitin and are suitable foruse in the process of the present invention. There are other naturalsources of chitin such as certain fungi, algae, yeast, insects, and someplants.

The removal of non-chitin rich organic material from shrimp shell wasteis accomplished by utilizing a mild sodium hydroxide pretreatmentsolution, for example, 1% w/v NaOH. This step is needed when there ispresent a quantity of non-chitin rich organic material, e.g., shrimpflesh present in shrimp shells disposed of by shrimp processors. A smallamount of proteins may also be removed from the surface of the shrimpshells in the pre-treatment step, aiding in the removal of proteins in alater deproteination step.

As shown in FIG. 1A, the shells are introduced as indicated by arrow 1into pre-treatment tank 10, in which the shells are immersed in a mildsodium hydroxide (NaOH) solution for two hours at room temperature, withagitation provided by a motorized mixer 12. The liquid-to-solids ratioin tank 10 may be six liters of the sodium hydroxide solution perkilogram of shrimp shell (6 L/kg, equivalent to 0.72 gal/lbm). Inlaboratory experiments, both bench-scale and large-scale, the shrimpshells were typically allowed to soak overnight in the mild sodiumhydroxide solution without agitation. Observation suggested that theshells were “clean” within two hours, and that the presence of agitationaids the process of removing the non-chitin rich organic material, thatis, the shrimp flesh. Accordingly, the pretreatment process time withmechanical agitation for shrimp shells may safely be about two hours atroom temperature.

After the pretreatment process is complete, the pretreated shrimp shellsare transferred to the pretreatment wash step by pumping the shrimpshells via line 14 to a static screen 16 located above pretreatment washtank 18. The spent sodium hydroxide liquid is separated from the shrimpshells by static screen 16 and pumped to a wastewater treatment system(not shown) via line 20. The shrimp shells fall into water in thepretreatment wash tank and are mechanically agitated by a motorizedmixer 22. The liquid-to-solids ratio in the pretreatment wash tank 18 isabout six liters per kilogram of treated shrimp shell (6 L/kg or 0.72gal/lbm). Once the shrimp shells have been added to the wash tank 18,the liquid is circulated through the static screen, where thepretreatment wash water is removed and sent to a wastewater treatmentsystem (not shown), while the shrimp shells fall back into the wash tank18. Simultaneously, fresh water is added to the wash tank 18 by meansnot shown, to maintain the liquid-to-solids ratio. This process isperformed for about one hour, after which time the water/shrimp shellmixture is sent to the demineralization tank 26, as described below.

Demineralization

Shrimp shells contain essentially three components: minerals, proteinsand chitin. The demineralization step removes the minerals in the shrimpshells using a mild hydrochloric acid solution, for example, 1 M HCl.

The pretreated shrimp shells are pumped via line 24 from thepretreatment wash tank 18 to a static screen 28 where the liquid isseparated from the clean shells and pumped via line 30 to a wastewatertreatment system (not shown). The shrimp shells fall from screen 28 intothe demineralization tank 26 in which water at ambient temperature isbeing agitated by motorized mixer 32. After all the shrimp shells havebeen added to the tank 26, an amount of concentrated HCl (22° Bée)needed to create a 1 M HCl demineralization solution in the tank 26 ismetered into the liquid over a period of twenty minutes. Metering theconcentrated HCl into the process prevents excessive foaming caused bythe release of carbon dioxide from the reaction between the acid and theminerals, the latter primarily comprising calcium carbonate. Thesolution is mixed for about one hour at room temperature, including thetime that the HCl is metered into the liquid. The liquid-to-solids ratioin demineralization tank 26 is about four liters per kilogram of cleanshell (4 L/kg or 0.48 gal/lbm). At the end of the treatment time, thedemineralized shrimp shells are pumped via line 34 to static screen 35mounted atop demineralization wash tank 36. The demineralization liquidis separated in static screen 35 from the demineralized shrimp shellsand the liquid is pumped via line 37 to a wastewater treatment system(not shown). The shrimp shells are deposited into demineralization washtank 36.

The demineralization wash step in wash tank 36 is performed in the samemanner as the pretreatment wash step, with the liquid-to-solids ratioset at four liters per kilogram of demineralized shell (4 L/kg or 0.48gal/lbm). Once the shells have been washed for one hour with agitationby motorized mixer 38, the water/demineralized shell mixture is pumpedto the deproteination step as described below.

Deproteination

The deproteination step removes the proteins from the shrimp shellsusing a mild sodium hydroxide solution, for example, about 5% w/v NaOH.Once the deproteination step is complete, the remaining component is thebiopolymer chitin.

The demineralized shrimp shells are pumped via line 40 from thedemineralization wash tank 36 to static screens 42 a, 42 b located atop,respectively, deproteination tanks 44 a, 44 b. The demineralizationliquid is separated from the demineralized shells in screens 42 a, 42 band pumped via lines 46 a, 46 b to a wastewater treatment system (notshown). The demineralized shells fall into the deproteination tanks 44a, 44 b in which a 5% w/v NaOH deproteination solution heated to about70° C. is being agitated by motorized mixers 43 a, 43 b. In the designof the process line and the determination of the process timetable, itis advantageous to have two process tanks for the deproteination stepinstead of one, as is the case in the other steps. The use of twodeproteination tanks allowed six batches of shrimp shells to beprocessed in a single day by a single line, thereby increasing thethroughput of one production line and reducing the number of productionlines needed, thereby resulting in lower capital costs. The solutionsare mixed for six hours in deproteination tanks 44 a, 44 b with thetemperature being maintained at about 70° C. The liquid-to-solids ratioin deproteination tanks 44 a, 44 b is about four liters per kilogram ofdemineralized shell (4 L/kg or 0.48 gal/lbm). Deproteination stepsperformed in the laboratory ranged in time from four to nineteen hours(overnight operation). Process times longer than four hours did not makea noticeable difference in the quality of the chitosan product of theprocess, but the process timing scheme benefited from a six-hourtreatment time in the deproteination step.

After the deproteination step treatment time ends, the resulting chitinis pumped from the deproteination tanks 44 a, 44 b via line 48 (FIG. 1A)to static screen 49 (FIG. 1B) in which liquid is separated from thechitin. As shown in FIG. 1B, the liquid is sent via line 51 to awastewater treatment system (not shown) and the chitin is deposited intothe deproteination wash tank 50 which is supplied with wash water bymeans not shown. The wash step is performed in the same manner asprevious wash steps at ambient temperature with agitation by motorizedmixer 52 and a liquid-to-solids ratio of about four liters per kilogramof chitin (4 L/kg or 0.48 gal/lbm). Once the chitin has been washed forone hour, the water/chitin mixture is pumped via line 54 to a staticscreen 56 set atop a simple belt press 58. The deproteination wash wateris separated from the chitin and discharged to drains via lines 56 a and58 a from, respectively, screen 56 and belt press 58. The chitin fallson the belt press 58, which presses excess water from the chitin. Thechitin is then transferred by screw auger 60 (or by a conveyor belt orany other suitable means) to a rotating dryer 62 which is rotated bydryer motor 64. The drying temperature is the same as that described inparagraph [0030] below, in connection with drying the chitosan product.The dryer 62 incorporates a return system 66 comprised of augers 68, 70(or screw conveyers or the like) and a reversible conveyer belt 72.Return system 66 may be operated to reintroduce partially dried chitinback into the dryer 62 one or more times in order to get as dry amaterial as is feasible. Adequate drying is important for chitin becausethe more moisture that is contained in the chitin, the greater thereduction in the concentration of the sodium hydroxide solution in thedeacetylation step, described below in connection with FIG. 2. Reducingthe sodium hydroxide concentration in the deacetylation stepconcomitantly reduces the effectiveness of the deacetylation process.

A hopper 74 is fed by dryer 62 to discharge dried chitin from dryer 62,the dried chitin being conveyed by an air conveyance pipe 76 (or anyother suitable means) to a storage tank (not shown) or directly to thechitosan production line illustrated in FIG. 2 and described below, orto other processing.

The Mathur/Narang article noted above suggests that the order of thedemineralization and deproteination steps can be interchanged dependingon the shells being processed. Laboratory experiments were conductedwith shrimp shells, with the deproteination preceding thedemineralization and vice versa. It was determined that better resultscome from performing the demineralization before the deproteinationstep. Without wishing to be bound thereby, it is believed that thereason for the better results obtained by performing demineralizationbefore deproteination may lie in the size of the molecules targeted inthe two steps. The minerals are much smaller molecules and more numerousthan the proteins; therefore, the hydrolysis of the proteins may be moreeasily achieved when the minerals are not present. That also applies toshells of other marine animals, including crustaceans.

Deacetylation

FIGS. 2A and 2B illustrate a production line for manufacturing chitosanfrom the chitin produced by the production line illustrated in FIGS. 1Aand 1B. As shown in FIG. 2A, a deacetylation step is employed to removethe acetyl group from N-acetylglucosamine (the chitin monomer) creatingan amine group, which results in d-glucosamine as the chitosan monomer,thus forming the biopolymer chitosan. The number of chitin monomersconverted, or the degree of deacetylation (expressed as a percentage),is a measure of the effectiveness of the deacetylation step. A highdegree of deacetylation is of course desirable.

Dry chitin obtained by the process described with reference to FIGS. 1Aand 1B is added via pipe 76 (FIG. 2A) to a concentrated sodium hydroxidedeacetylation solution of about 50% w/w NaOH at a temperature of about100° C. in deacetylation tank 78, with agitation by motorized mixer 80.The chitin feed to deacetylation tank 78 has been dried to the extentfeasible by removing as much residual water from it, in order to reducethe amount of water, and therefore reduce the amount of dilution of theconcentrated sodium hydroxide in deacetylation tank 78. Removal ofresidual water may be carried out by any suitable means, for example,pressing, heating at a maximum temperature of 65° C., or a combinationof pressing and heating. The liquid-to-solids ratio in deacetylationtank 78 is about fifty liters per kilogram of chitin (50 L/kg or 5.99gal/lbm). This step is performed for about three hours. Once thetreatment time ends, the chitosan/sodium hydroxide deacetylationsolution is pumped via line 82 to a static screen 84 set atop a simplebelt press 86. The sodium hydroxide deacetylation solution is separatedfrom the chitosan in static screen 84 and pumped via line 88 into asurge tank 90, while the chitosan falls onto the belt press 86, whichpresses out excess sodium hydroxide solution, which is also sent vialines 88 a and 88 to the surge tank 90. Liquid in surge tank 90 ispumped via line 92 and filter system 94 to used sodium hydroxide storagetank 96. The sodium hydroxide in tank 96 is re-used by being pumpedthrough line 98 to deacetylation tank 78.

The chitosan is then transferred from belt press 86 via horizontal auger100 and vertical auger 102 to the deacetylation wash tank 104.Deacetylation wash tank 104 performs in a manner similar to the washtanks of the previous steps. Once the chitosan has been washed indeacetylation wash water for about one hour at ambient temperature withagitation by motorized mixer 106, it is pumped through line 105 to ascreen 108 set atop a simple belt press 110. The water is separated fromthe chitosan, and the separated water is sent via line 107 to a drain.The chitosan falls on the belt press 110, which presses water from thechitosan and this used deacetylation wash water is sent to a drain vialine 109. The chitosan is then transferred via an auger 60′ (FIGS. 2Aand 2B) to a dryer 62′ (FIG. 2B).

As shown in FIG. 2B, auger 60′ is part of a return system 66′ which issubstantially the same as return system 66 of FIG. 1B and operates inthe same manner as return system 66, that is, return system 66′ iscapable of returning chitosan removed from dryer 62′ back to dryer 62′in order to subject the chitosan to repeated drying cycles. Adequatedrying is important inas-much as a low moisture content in the chitosanis desirable for the final product, so the drying should be as completeas possible. The drying temperature for the chitosan should not,however, exceed about 65° C., as higher temperatures cause the chitosanto turn from white to pale yellow. The return system 66′ helps thechitosan drying process by providing two or more passes through thelow-temperature dryer, which, for example, may be operated at atemperature of from about 37° C. to about 60° C. to 65° C. The upperlimit on temperature may be somewhat below 65° C. to insure that thereis no yellowing of the chitosan, especially if temperature variationsmay occur. Therefore, the upper limit may be held to, for example, about60° C., 61° C., 62° C., 63° C. or 64° C., or even lower.

The components of return system 66′ are numbered identically to those ofreturn system 66 except for the addition of a prime indicator theretoand as they function identically to the components of return system 66is it not necessary to provide a detailed description of system 66′ andits operation. Air conveyance pipe 76′ transfers the dried chitosan fromreturn system 66′ to a packaging system 112 wherein the chitosan, whichis in flake form, is appropriately packaged for shipment. Obviously,instead of being packaged, some or all of the chitosan may be conveyedby pipe 76′ directly to another production line for use or for furthertreatment. Equally obviously, other treatment equipment (not shown) maybe introduced into the production line of FIGS. 1A and 1B and/or FIGS.2A and 2B at appropriate locations for other treatment by known methodsfor purification, grinding, etc., of the chitosan as described below.

Referring now to FIGS. 3, 3A and 3B, there is shown a process tank 114which is typical of the screen-equipped tanks of FIGS. 1 and 2. Processtank 114 comprises a tank body 116 having mounted thereon a motorizedmixer 118 which is driven by a motor 118 a (FIGS. 3A and 3B). Motorizedmixer 118 is supported atop tank body 116 by a cradle 120. Tank body 116is supported by a plurality of stanchions 122, 124. A screen 126 is alsocarried by cradle 120 and has a screen inlet line 128 and a screenoutlet line 130 connected thereto for introducing process material intoscreen 126 via inlet line 128 and withdrawing liquid therefrom viaoutlet line 130.

Tank body 116 may of course be made of any suitable material such assteel or fiberglass. For process tanks which are operated at elevatedtemperatures, a tank construction substantially similar to thatillustrated in FIGS. 3, 3A and 3B is utilized, except that the tank hasa steam jacket disposed around the exterior thereof and is equipped witha hinged lid to retain heat within the tank, the lid permitting accessto the tank interior. Contents of tank 114 may be withdrawn via a tankoutlet line 132, a pump 134 and a pump outlet line 136.

In general, treatment of the waste streams from the chitosanmanufacturing process is undertaken in the same manner as for anysimilar aqueous waste streams, focusing on those contaminants thatexceed local discharge requirements. In order to more efficiently treatthe wastewater, the waste streams from the pretreatment, pretreatmentwash, deproteination, deproteination wash and the deacetylation wash aremixed together to create a single stream which will have a pH around 13(basic waste). The waste streams from the demineralization anddemineralization wash are combined, creating an acidic waste which isthen added to the basic waste. As the acidic waste is added to the basicwaste, calcium hydroxide (Ca(OH)₂) precipitates out of solution,resulting in a reduction in the overall pH of the wastewater toapproximately 10 and clarifying the wastewater by an entrapment ofsuspended materials by the precipitating calcium hydroxide. Removal ofthe calcium hydroxide precipitate leaves a translucent, pale yellowwastewater that is then neutralized and treated to meet dischargerequirements.

EXAMPLE 1

Chitosan material derived from shrimp shells in accordance with theabove-described process was characterized to determine the material'scharacteristics of degree of deacetylation (“DDA” in Table I below),molecular weight, moisture content, and residual protein and ashcontent. Degree of deacetylation (“DDA”) was determined based onacid-base titration using methyl orange as pH indicator (Broussignac P.Chim. Ind. Genie. Chim. 1968, 99:1241; Domszy J G, Roberts G A F.Makromol. Chem. 1985, 186:1671). Molecular weight of the material wasdetermined based on viscometry (Wang W, Bo S, Li S, Qin W. Int J BiolMacromol, 13:281-285, 1991) and by gel permeation chromatography (GPC)using dextran as standard (Ratajska M, Wisniewska-Wrona M, Strobin G,Struszczyk H, Boryniec S, Ciechanska D. Fibers & Textiles in EasternEurope, 11:59-63, 2003). Moisture content was determined according toASTM F2103-01 Standard Guide for Characterization and Testing ofChitosan Salts as Starting Materials Intended for Use in Biomedical andTissue-Engineering Medical Product Applications. Residual proteincontent was determined via the bicinchoninic acid (“BCA”) assayutilizing a BCA Protein Assay Kit manufactured by Pierce Biotechnologyof Rockford, Ill. Residual ash content was determined via combustion at550° C. as described in an article by Tingda Jiang, CHITOSAN, Chemicalindustry press, Beijing, China. 2001, p 108.

Results

The results of the analyses of a chitosan product produced by the abovedescribed method and designated “Sample A” are presented in tables below(Table II contains the results of the GPC molecular weight analysis).Sample A was produced by a “large-scale” chitosan manufacturing runperformed with 44 kilograms of shrimp shell. Sample A was pretreatedovernight, demineralized for 1.25 hours in a 0.909 M HCl solution,washed, deproteinated overnight at an average temperature of 72° C.,washed, deacetylated for 3 hours in 50% NaOH solution at an averagetemperature of 111° C., washed and dried at 65° C. The resultingchitosan was white flakes. Sample A was not purified or ground into apowder. Values are expressed as mean±standard deviation of number (n) ofsamples measured as indicated. It is noted that residual protein contentwas below the detection limit of the above-mentioned BCA assay.

TABLE I Residual Protein Mois- Residual Sam- DDA M_(v) ⁽²⁾ (valuesarebelow ture Ash ple (%)⁽¹⁾ (Daltons*10⁶) detection limit) (%) (%) Sam-82.85 ± 1.40 <0.19% or 9.14 ± 0.097 ± ple A 0.40 <1.94 mg/g 0.22 0.003(n = 3) Chitosan (n = 3) (n = 3) ⁽¹⁾Degree of deacetylation is thepercentage obtained by dividing the number of acetyl groups re-moved bythe number of acetyl groups originally present, and multiplying by onehundred. ⁽²⁾Molecular weight (viscosity-average) value.

TABLE II Polydispersity Sample M_(W) ⁽³⁾ M_(N) ⁽⁴⁾ M_(V) Index⁽⁵⁾ SampleA 104620.29 662.21 103592.16 157.99 ⁽³⁾Molecular weight (weight-average)value. ⁽⁴⁾Molecular weight (number average) value. ⁽⁵⁾The ratio ofweight-average molecular weight to number average molecular weight.

The chitosan characteristics achieved with the above process aresummarized below. The above-described process was intended to be capableof manufacturing a chitosan that would qualify as medical-grade quality.While there seems to be no set standard regarding the characteristics ofmedical-grade quality chitosan, information obtained from the internetregarding pharmaceutical grade chitosan suggests that the materialcharacteristics shown in Table III under the heading “Value”, aretypical for medical-grade quality chitosan. As Table II shows, thereported values for the chitosan of Sample A exceeds the requirementsfor medical-grade quality chitosan.

TABLE III Reported Value for Characteristic Value Chitosan Sample ADegree of Deacetylation 80% or greater 82.85 ± 0.40%  Protein Contentless than 0.3% less than 0.19% Ash Content less than 0.2% 0.097 ± 0.003%Moisture Content less than 10% 9.14 ± 0.22%

The above characteristics of the chitosan produced by theabove-described process serve as an excellent starting point for allgrades of chitosan, up to and including medical-grade. In addition tothe process steps described above, purification steps to remove foreignparticles, heavy metals, arsenic, mercury, lead and microbiologicalcontaminants may be carried out by methods known to the art. A grindingstep to produce a chitosan powder, which appears to be a common form ofhigher grades of chitosan, may also be carried out.

Utilizing the tank/screen/mixer arrangement for all the steps in theprocess allows the overall process to be scaled to handle practicallyany amount of shrimp shells, or other chitosan-containing raw material,per day. The designer has the option of increasing the size of thetanks, or utilizing multiple lines to achieve the desired shrimp shellor other chitosan-containing raw material processing rate. The percentloss of material in each step has been experimentally measured, so byknowing the amount of shrimp shell to be processed in a single batch,the designer can specify the water and chemical requirements as well asthe tank sizes for each step. These values can be programmed into theprocess control system along with the processing conditions and timingsso that the chitosan produced is consistent from batch to batch.

The above-described basic four-step process produces a consistent highquality chitosan of at least industrial grade which may serve as theprecursor to the processing of higher grades of chitosan with theaddition of known purification and grinding steps. The process is easilyscalable knowing the amount of shrimp shell or other chitosan-containingraw material to be processed, allowing the process to be tailored to anysituation where shrimp shell waste or other chitosan-containing rawmaterial is available.

What is claimed is:
 1. A process for the manufacture of chitosan from anaturally occurring chitin source consists essentially of the followingsteps: (a) a naturally occurring chitin source is demineralized byimmersing it in a demineralization (“DMIN”) hydrochloric acid solutionof from about 0.5 to about 2 M at a temperature of from about 20° C. toabout 30° C. and for a DMIN time period of from about 0.5 to about 2hours to demineralize the chitin source, and then separating theresulting demineralized chitin source from the acid solution, washingthe chitin source in a DMIN wash water for a DMIN wash period of fromabout 0.5 to about 2 hours to remove the hydrochloric acid and calciumsalts therefrom, and then separating the demineralized chitin sourcefrom the DMIN wash water; (b) subjecting the demineralized chitin sourceto deproteination (“DPRO”) by treating the demineralized chitin sourcein a DPRO sodium hydroxide solution containing from about 1% to about10% w/w NaOH for a DPRO time period of from about 4 to about 24 hoursand at a temperature of from about 60° C. to about 80° C. todeproteinize the demineralized chitin source, and then separating theresulting demineralized and deproteinized chitin source from thedeproteination sodium hydroxide solution, washing the separateddemineralized and deproteinized chitin source in a DPRO wash water for aDPRO wash period of from about 0.5 to about 2 hours to remove the sodiumhydroxide from the demineralized and deproteinized chitin source, andthen separating the demineralized and deproteinized chitin source fromthe deproteination wash water; (c) separating residual water from thechitin source obtained in step (b); (d) immersing the chitin sourceobtained from step (b) into a sodium hydroxide deacetylation (“DEAC”)solution containing from about 40% to about 50% w/w NaOH and carryingout deacetylation at a temperature of from about 90° C. to about 110° C.and for a DEAC time period of from about 1 to about 3 hours to convertacetyl groups of the chitin source obtained from step (c) to aminegroups, to thereby form a chitosan biopolymer having d-glucosamine asthe monomer of the chitin biopolymer, then separating the resultingchitosan biopolymer from the DEAC solution and washing the separatedchitosan biopolymer in a DEAC wash water for a DEAC wash periodsufficient to remove sodium hydroxide from the chitosan polymer, andthen separating the chitosan biopolymer from the DEAC wash water; and(e) residual water is then separated from the chitosan biopolymer whichis then dried in air at a temperature of not more than about 65° C. fora drying time period sufficient to reduce the moisture content of thechitosan biopolymer to below about 10% by weight to provide amedical-grade quality chitosan.
 2. The process of claim 1 wherein step(e) is carried out under conditions comprising that the temperature isfrom about 50° C. to about 65° C. and the drying period is from about 2to about 5 hours.
 3. The process of claim 1 wherein: step (a) is carriedout under conditions comprising that the hydrochloric acid solution isfrom about 0.9 to about 1.1 M, the temperature is from about 22° C. toabout 26° C., the DMIN time period is from about 0.75 to about 1.25hours, and the DMIN wash period is from about 0.9 to about 1.1 hours;step (b) is carried out under conditions comprising that the sodiumhydroxide solution contains from about 4% to about 6% w/w NaOH, thetemperature is from about 70° C. to about 75° C., the deproteinationperiod is from about 4 to about 6 hours, and the DPRT wash period isfrom about 0.9 to about 1.1 hours; and step (d) is carried out underconditions comprising that the sodium hydroxide solution contains fromabout 45% to about 50% w/w NaOH at a temperature of from about 100° C.to about 110° C. and the deacetylation wash period is from about 0.9 toabout 1.1 hours.